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PubMed2005

Dynamic receptor-based pharmacophore model development and its application in designing novel HIV-1 integrase inhibitors.

We present here a dynamic receptor-based pharmacophore model representing the complementary features of the active site region of HIV-1 integrase (IN), which was developed from a series of representative conformations of IN. Conformations of IN were sampled through a molecular dynamics study of the catalytic domain of an IN monomer, and an ensemble of representative IN structures were collected via a probability-based representative conformer sampling method that considers both the potential energy and the structural similarity of the protein conformations. The dynamic pharmacophore model was validated by a set of 128 known inhibitors, and the results showed that over 72% of the active inhibitors (IC(50) lower than 20 microM) could be successfully identified by the dynamic model. Therefore, we screened our in-house database of commercially available compounds against this model and successfully identified a set of structurally novel IN inhibitors. Compounds 7 and 18 with IC(50)s of 8 microM and 15 microM, respectively, against the strand transfer reaction were the most potent. Moreover, 7, 8 and 20 showed a 5-fold selectivity for the strand transfer reaction over 3'-processing.

Binding Sites↗
PubMed2000

Minimal core domain of HIV-1 integrase for biological activity.

The human immunodeficiency virus type-1 (HIV-1) integrase (IN) mediates insertion of viral DNA into human DNA, which is an essential step in the viral life cycle. In order to study minimal core domain in HIV-1 IN protein, we constructed nine deletion mutants by using PCR amplification. The constructs were expressed in Escherichia coli, and the proteins were subsequently purified and analyzed in terms of biological activity such as enzymatic and DNA-binding activities. The mutant INs with an N-terminal or C-terminal deletion showed strong disintegration activity though they failed to show endonucleolytic and strand transfer activities, indicating that the disintegration reaction does not require the fine structure of the HIV-1 IN protein. In the DNA-binding analysis using gel mobility shift assay and UV cross-linking method, it was found that both the central and C-terminal domains are essential for proper DNA-IN protein interaction although the central or C-terminal domain alone was able to be in close contact with DNA substrate. Therefore, our results suggest that the C-terminal domain act as a DNA-holding motive, which leads to proper interaction for enzymatic reaction between the IN protein and DNA.

Binding Sites↗
PubMed2006

Quantitative structure-activity relationship studies of HIV-1 integrase inhibition. 1. GETAWAY descriptors.

The GEometry, Topology, and Atom-Weights AssemblY (GETAWAY) approach has been applied to the study of the HIV-1 integrase inhibition of 172 compounds that belong to 11 different chemistry families. A model able to describe more than 68.5% of the variance in the experimental activity was developed with the use of the mentioned approach. In contrast, none of the five different approaches, including the use of Randić Molecular Profiles, Geometrical, RDF, 3D-MORSE and WHIM descriptors was able to explain more than 62.4% of the variance in the mentioned property with the same number of variables in the equation. Finally, after extracting five compounds considered by us as outliers the model was able to describe more than 72.5% of the variance in the experimental activity.

Cluster Analysis↗
PubMed2000

The plant ribosome inactivating proteins luffin and saporin are potent inhibitors of HIV-1 integrase.

The ribosome inactivating proteins (RIPs) are a group of proteins that are able to inactivate eukaryotic protein synthesis by attacking the 28S ribosomal RNA. Recent studies have shown that some RIPs possess strong anti-human immunodeficiency virus (HIV) activity. In this study, several common plant RIPs including agrostin, gelonin, luffin, alpha-momorcharin, beta-momorcharin, saporin and trichosanthin were examined for the ability to interfere with HIV-1 replication in a variety of mechanistic assays in vitro. These assays included the CD4/gp120 interaction assay, HIV-1 reverse transcriptase (RT) assay, HIV-1 protease assay and HIV-1 integrase assay. At the concentration of 100 nM, all RIPs appeared to enhance the CD4/gp120 interaction by about 50%. These RIPs exhibited a very weak suppressive effect on HIV-1 RT and on HIV-1 protease. In contrast, with the exception of agrostin, all the RIPs tested could strongly inhibit HIV-1 integrase, the extent of inhibition ranging from 26.1 to 96.3% in an ELISA-based assay. Two RIPs, saporin and luffin, which licited over 90% inhibition in the ELISA-based assay, were further characterized in a radiometric assay. Both of these two RIPs evoked a strong dose-dependent inhibition in the 3'-end processing and strand-transfer activities of integrase. The results from this study suggest that the anti-HIV property of RIPs may be due to inhibition of HIV-1 integrase.

Anti-HIV Agents↗
PubMed2004

HIV-1 integrase inhibitors: a decade of research and two drugs in clinical trial.

AIDS is currently treated with a combination therapy of reverse transcriptase and protease inhibitors. Recently, the FDA approved a drug targeting HIV-1 entry into cells. There are currently no FDA approved drugs targeting HIV-1 integrase, though many scientists and drug companies are actively in pursuit of clinically useful integrase inhibitors. The objective of this review is to provide an update on integrase inhibitors reported in the last two years, including two novel inhibitors in early clinical trials, recently developed hydroxylated aromatics, natural products, peptide, antibody and oligonucleotide inhibitors. Additionally, the proposed mechanism of diketo acid inhibition is reviewed.

Acetoacetates↗
PubMed2000

Chemical and enzymatic modifications of integric acid and HIV-1 integrase inhibitory activity.

Integric acid (1), an acyl eremophilane sesquiterpenoid, was identified as an inhibitor of HIV-1 integrase, the enzyme responsible for provirus entry into the host cell nucleus and integration in to the host genome. Chemical and enzymatic modification of integric acid led to the preparation of several selective chemical derivatives of integric acid. Preparation, HIV-1 inhibitory activity, and the structure-activity relationship against coupled and strand transfer assays are described. It appears that most of the groups present in the natural product are required for inhibition of HIV-1 integrase strand transfer activity. In contrast, inhibition of 3' processing activity is less stringent suggesting distinct SAR for the two integrase reactions.

Amides↗
PubMed2000

Active site binding modes of HIV-1 integrase inhibitors.

Using the crystal structure of the first complex of the HIV-1 integrase catalytic core domain with an inhibitor bound to the active site, structural models for the interaction of various inhibitors with integrase were generated by computational docking. For the compound of the crystallographic study, binding modes unaffected by crystal packing have recently been proposed. Although a large search region was used for the docking simulations, the ligands investigated here are found to bind preferably in similar ways close to the active site. The binding site is formed by residues 64-67, 116, 148, 151-152, 155-156, and 159, as well as by residue 92 in case of the largest ligand of the series. The coherent picture of possible interactions of small-molecule inhibitors at the active site provides an improved basis for structure-based ligand design. The recurring motif of tight interaction with the two lysine residues 156 and 159 is suggested to be of prime importance.

Anthraquinones↗
PubMed2000

Synthesis and HIV-1 integrase inhibitory activities of caffeoylglucosides.

Caffeoylglucosides, which have a glucose ring as a central linker, were synthesized from methyl D-glucosides, and their anti-HIV-1 activities were tested. Among them, four dicaffeoylglucosides (IC50 = 29.1+/-35.1 microM), 6a, 6b, 9b and 10b, showed HIV-1 integrase inhibitory activity as potent as L-chicoric acid.

Anti-HIV Agents↗
PubMed1997

Critical contacts between HIV-1 integrase and viral DNA identified by structure-based analysis and photo-crosslinking.

Analysis of the crystal structure of HIV-1 integrase reveals a cluster of lysine residues near the active site. Using site-directed mutagenesis and photo-crosslinking we find that Lys156 and Lys159 are critical for the functional interaction of integrase with viral DNA. Mutation of Lys156 or Lys159 to glutamate led to a loss of both 3' processing and strand transfer activities in vitro while maintaining the ability to interact with nonspecific DNA and support disintegration. However, mutation of both residues to glutamate produced a synergistic effect eliminating nearly all nonspecific DNA interaction and disintegration activity. In addition, virus containing either of these changes was replication-defective at the step of integration. Photo-crosslinking, using 5-iododeoxyuracil-substituted oligonucleotides, suggests that Lys159 interacts at the N7 position of the conserved deoxyadenosine adjacent to the scissile phosphodiester bond of viral DNA. Sequence conservation throughout retroviral integrases and certain bacterial transposases (e.g. Tn10/IS10) supports the premise that within those families of polynucleotidyl transferases, these residues are strategic for DNA interaction.

Amino Acid Sequence↗
PubMed2004

Rapid, high-throughput purification of HIV-1 integrase using microtiter plate technology.

The human immunodeficiency virus type-1 (HIV-1) integrase (IN) catalyzes the insertion of the retroviral genome into the chromosome of an infected host cell. HIV-1 IN was expressed as a N-terminal hexa-histidine fusion in Escherichia coli. A high-throughput purification strategy was developed using denaturing methods for the initial protein extraction, followed by a one-step nickel-chelating chromatography purification and step-wise refolding. IN was routinely greater than 90% pure with yields exceeding 14 microg of purified IN per ml of E. coli culture. In vitro 3' processing and strand transfer assays showed the enzyme preparations to be highly active. The specific activity of the purified IN was 2.65 pmol/h/microg IN, which is very similar to the activity of IN routinely produced by large-scale column chromatographic methods. This high-throughput platform should be of general utility to those interested in defining the structure-function relationship of proteins and enzymes.

Cell Culture Techniques↗
PubMed2002

Interaction of HIV-1 integrase with DNA repair protein hRad18.

We have previously shown that human immunodeficiency virus-1 (HIV-1) integrase is an unstable protein and a substrate for the N-end rule degradation pathway. This degradation pathway shares its ubiquitin-conjugating enzyme, Rad6, with the post-replication/translesion DNA repair pathway. Because DNA repair is thought to play an essential role in HIV-1 integration, we investigated whether other molecules of this DNA repair pathway could interact with integrase. We observed that co-expression of human Rad18 induced the accumulation of an otherwise unstable form of HIV-1 integrase. This accumulation occurred even though hRAD18 possesses a RING finger domain, a structure that is generally associated with E3 ubiquitin ligase function and protein degradation. Evidence for an interaction between integrase and hRad18 was obtained through reciprocal co-immunoprecipitation. Moreover we found that a 162-residue region of hRad18 (amino acids 65-226) was sufficient for both integrase stabilization and interaction. Finally, we observed that HIV-1 integrase co-localized with hRad18 in nuclear structures in a subpopulation of co-transfected cells. Taken together, these findings identify hRad18 as a novel interacting partner of HIV-1 integrase and suggest a role for post-replication/translesion DNA repair in the retroviral integration process.

Blotting, Western↗
PubMed2005

Synthesis and HIV-1 integrase inhibitory activities of caffeic acid dimers derived from Salvia officinalis.

The synthesis of two caffeoyl-coumarin conjugates, derived from sagecoumarin, has been accomplished, starting from ferulic acid, isoferulic acid and sesamol. Both compounds exhibited potent inhibitory activities at micromolar concentrations against HIV-1 integrase in 3'-end processing reaction but were less effective against HIV-1 replication in a single-round infection assay of HeLa-beta-gal-CD4+ cells.

Caffeic Acids↗
PubMed2005

HIV-1 integrase crosslinked oligomers are active in vitro.

The oligomeric state of active human immunodeficiency virus type 1 (HIV-1) integrase (IN) has not been clearly elucidated. We analyzed the activity of the different purified oligomeric forms of recombinant IN obtained after stabilization by platinum crosslinking. The crosslinked tetramer isolated by gel chromatography was able to catalyze the full-site integration of the two viral LTR ends into a target DNA in vitro, whereas the isolated dimeric form of the enzyme was involved in the processing and integration of only one viral end. Accurate concerted integration by IN tetramers was confirmed by cloning and sequencing. Kinetic studies of DNA-integrase complexes led us to propose a model explaining the formation of an active complex. Our data suggest that the tetrameric IN bound to the viral DNA ends is the minimal complex involved in the concerted integration of both LTRs and should be the oligomeric form targeted by future inhibitors.

Cross-Linking Reagents↗
PubMed2000

Cyclodidemniserinol trisulfate, a sulfated serinolipid from the Palauan ascidian Didemnum guttatum that inhibits HIV-1 integrase.

[structure--see text] Bioassay-guided fractionation of extracts of the Palauan ascidian Didemnum guttatum led to the isolation of cyclodidemniserinol trisulfate (1) as an inhibitor of HIV-1 integrase, which is an attractive target for anti-retroviral chemotherapy. The structure of cyclodidemniserinol trisulfate (1), the stereochemistry of which was only partially determined, was elucidated by interpretation of NMR and mass spectral data.

Animals↗
PubMed2004

HIV-1 integrase complexes with DNA dissociate in the presence of short oligonucleotides conjugated to acridine.

The human immunodeficiency virus type 1 (HIV-1) integrase is an essential enzyme in the life cycle of the virus and is therefore an attractive target for the development of new antiviral drugs. Among them, inhibitors which are capable of targeting the preassembled integrase/DNA complex are of particular interest, because they could suppress integrase activity in the context of the HIV-1 preintegration complex. Here, we study the mechanism of action of 11-mer oligonucleotides, which are efficient inhibitors of the catalytic activity of integrase, provided that they are conjugated to a hydrophobic compound, acridine. To understand the mechanism of the conjugate inhibitory action, we used a steady-state fluorescence anisotropy assay, which allowed us to study the stability of the integrase/DNA complex in various conditions. We found that oligonucleotide-acridine conjugates induced the efficient dissociation of preassembled integrase/DNA complexes. The simultaneous presence of both acridine and an oligonucleotidic moiety is required for the inhibitory activity of conjugates. However, the dissociation effect is not dependent on the oligonucleotide sequence. Finally, our results suggest that the conjugates bind directly to integrase within its complex with DNA at a site different from the viral DNA binding site.

Acridines↗
PubMed2002

Structure activity of 3-aryl-1,3-diketo-containing compounds as HIV-1 integrase inhibitors.

The 4-aryl-2-hydroxy-4-oxo-2-butenoic acids and their isosteric tetrazoles are among an emerging class of aryl beta-diketo (ADK)-based agents which exhibit potent inhibition of HIV-1 integrase (IN)-catalyzed strand transfer (ST) processes, while having much reduced potencies against 3'-processing (3'-P) reactions. In the current study, L-708,906 (10e) and 5CITEP (13b), which are two examples of ADK inhibitors that have been reported by Merck and Shionogi pharmaceutical companies, served as model ADK leads. Structural variations to both the "left" and "right" sides of these molecules were made in order to examine effects on HIV-1 integrase inhibitory potencies. It was found that a variety of groups could be introduced onto the left side aryl ring with maintenance of good ST inhibitory potency. However, introduction of carboxylic acid-containing substituents onto the left side aryl ring enhanced 3'-P inhibitory potency and reduced selectivity toward ST reactions. Although both L-708,906 and 5CITEP show potent inhibition of IN in biochemical assays, there is a disparity of antiviral activity in cellular assays using HIV-1-infected cells. Neither 5CITEP nor any other of the indolyl-containing inhibitors exhibit significant antiviral effects in cellular systems. Alternatively, consistent with literature reports, L-708,906 does provide antiviral protection at low micromolar concentrations. Interestingly, several analogues of L-708,906 with varied substituents on the left side aryl ring, while having good inhibitory potencies against IN in extracellular assays, are not antiviral in whole-cell systems.

Acetoacetates↗
PubMed2000

Inactivation of the SNF5 transcription factor gene abolishes the lethal phenotype induced by the expression of HIV-1 integrase in yeast.

The ubiquitous human transcription factor Ini1 has been shown to interact with HIV-1 integrase (IN) and to stimulate in vitro the reactions catalyzed by this enzyme. We have previously used a yeast model to study the effect of HIV-1 IN expression (Caumont, A.B., Jamieson, G.A., Pichuantes, S., Nguyen, A.T., Litvak, S., Dupont, C. -H., 1996. Expression of functional HIV-1 integrase in the yeast Saccharomyces cerevisiae leads to the emergence of a lethal phenotype: potential use for inhibitor screening. Curr. Genet. 29, 503-510). Here, we describe the effect of the inactivation of the gene encoding for SNF5, a yeast transcription factor homologous to Ini1, on the lethality induced by the expression of HIV-1 IN in yeast. We observed that the retroviral IN was unable to perform its lethal activity in cells where the SNF5 gene has been disrupted, suggesting that SNF5 may play a role in the lethal effect induced by IN in yeast. SNF5 inactivation affects neither yeast viability nor expression of HIV-1 IN. Given the homology between SNF5 and its human counterpart Ini1, our results suggest that this factor may be important for IN activity in infected cells. Moreover, given the important role proposed for this transcription factor in the integration step and the fact that it is dispensable for cell viability, the interaction between Ini1/ySNF5 and HIV-1 IN should become a potential target in the search for new antiretroviral agents.

Amino Acid Sequence↗